Animal feed with low phytic acid, oil burdened and protein laden grain

ABSTRACT

The present invention provides grain, seed, feed made from the grain or seed, pet food made from the grain, and food products made from the grain. The grain may be maize grain with the following characteristics: oil burdened, elevated protein content, and low phytate levels. The combination of oil burdened, protein laden, and decreased phytate characteristics in grain makes a grain that provides more calories, protein and phosphorus and other nutrients to the feeding animal. Pet foods, and animal feeds and corn food products made of the present invention will provide increased nutrition because of the increased bioavailability of the components of the grain.

The present invention is based on and claims benefit to U.S. ProvisionalApplication Nos. 60/051,854 and 60/051,855, filed Jul. 7, 1997, theentire contents of which are incorporated herein by reference.

FIELD OF INVENTION

Broadly, the present invention relates to grain having increased oil andincreased protein and amino acids, increased total phosphorusbioavailability and decreased phytic acid. This grain can be used asfeedstuffs for animals. More particularly, this invention relates tograin based feed that provide improved animal nutrition, and reduces theenvironmental impact of animal production.

BACKGROUND

Over the last fifty years, approaches toward providing animal nutritionhave changed. No longer are the animals fed whatever grain or forage maybe available. Instead, the diets of animals are closely monitored fortotal nutrition value, and for cost. The animal on the diet ismonitored, for quality and performance characteristics, and for theenvironmental impact of the waste from the animal. The informationgathered is employed to adjust the feed to increase nutrition value ofthe feed and the animal performance characteristics while decreasing thecost and environmental impact.

Cereals and fats are used in feeding programs for nonruminants such asswine and poultry to provide a nutritional source of calories. The ratioof cereals to supplements, such as vitamins, minerals and fats, havechanged across years in an attempt to maximize feeding efficiency of theanimals. The feeding efficiency (the feed conversion ratio) or how muchfeed is required to produce one pound of animal weight is determined bya combination of matching the genetic potential of the animal, and thenutrients supplied to the animal. As the feed conversion ratio has risendue to genetic enhancements, the mineral nutrient requirements in thefeed have risen to assure a complete and heathy diet.

Since an animal's ability to feed limits the amount of nutrients andcalories it can consume, the feed industry has had to develop ways tomake feeds that are more highly caloric. To increase the caloric densityof the feed, producers have added fat to the feed. Fat has often beenadded to the feed in the form of a liquid. Fat has the advantage ofsupplying calories to each mouthful of feed. However, adding fat to feedhas some disadvantages such as costs, added labor and technicaldifficulties with automatic feeding systems. Additionally, the fat isoften of poor quality, thus reducing the overall quality of the feed.

To reduce the use of liquid fat in feeds, the industry has triedincreasing the oil content of the grain used in the feeds. The Dupontcompany has developed and commercialized high oil corn as a method forincreasing the oil content of feed. Other companies have developed cornthat has more oil than no.2 yellow dent corn but less than Dupont's highoil corn. High oil and elevated oil corn is herein alternativelyreferred to as oil burdened corn. This extra oil in the corn reduces andmay eliminate the need for the addition of the liquid fat to the feed.

Traditionally, oil burdened corn has been thought to contain increasedlevel of phytic acid, as compared with levels in No. 2 yellow dent corn.Raboy et al (Journal of Heredity 1989: 80: 311-315) have reportedhowever, that there is an apparent negative relationship betweenselection for oil and total phytic acid, phytic acid phosphorus andphosphorus per kernel, per germ and per endosperm of Illinois High Oiland Low Oil lines, as opposed to the previously expected apparentpositive relationship on a concentration basis (i.e., mg constituent perg kernel, germ or endosperm). Raboy explains that the discrepancybetween total contents per organ and concentration per organ resultsfrom the large divergence in organ dry weights exhibited between theIllinois High Oil (IHO) and Illinois Low Oil (ILO) seed used in hisstudy; IHO germ being about twice the dry weight of ILO germ and ILOendosperm having nearly three times the dry weight of IHO endosperm. Incontrast to this trend for high oil being linked to lower phytic acid,Raboy also reports a consistent positive relationship between increasingprotein selection and increasing amounts of phytic acid, phytic acidphosphorus and phosphorus. Thus, there is an apparent positiverelationship between selection for protein and total phytic acid, phyticacid phosphorus and phosphorus per kernel, per germ and per endosperm ofillinois high protein and low protein lines. This was maintained evenwhen the data are expressed on a concentration basis (i.e., mgconstituent per g kernel, germ or endosperm). Thus selection for proteinand oil appears to divergently affect phytate content in seed.

As reports suggest an average increase of 0.38% protein with each 1%increase in oil (Han Y. Et al., 1987 Poultry Science 66:103-111;Keshararz, Poultry Pointers, pp 6-7), it is uncertain from the artwhether grains containing high oil, high protein and low phytic acidcould be produced (Brewer, “Optimum® High Oil Corn Improves PoultryRations” Poultry Digest, February/March 1998 pp 30-31). Brewer statesthat while high oil corn is available as of 1998, varieties which arehigh in oil, high in protein and high in digestible phosphorus (i.e.,low in phytic acid phosphorus), have yet to be developed.

The concentration of phytic acid in grain-based diets has long been ofconcern to humans and animal nutritionists, because evidence has shownthat phytic acid acts to form insoluble salts with nutritionallyimportant minerals that subsequently are not absorbed in the intestine.Phytic acid (myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate)) isa form of phosphorus (P) in seeds which is stored in the form of phytatesalts. Phytate salts have a negative nutritional impact on the animalbecause phosphorus bound to phytate is not available to the animal as asource of nutrition. Moreover, the animal does not retain the mineralssuch as Ca, Zn and the like and these needed minerals are excreted.Finally, the animal waste contain phytate P which then contributes tothe surface and ground water pollution. If the grain is used for millingpurposes then the milling by-products contain phytate P which thencontributes to the surface and ground water pollution.

Swine, for example, lack the digestive enzyme (phytase) required tocleave the phosphorus from the phytate molecule and thus can not readilyuse phytate-phosphorus. Increasing the availability of phosphorus byelimination of the phytate salts binding the phosphorus would enable areduction in dietary total phosphorus content without jeopardizing theanimal's health or production performance. Increasing thebioavailability of phosphorus results in a lower phosphorus content inthe swine wastes, which is environmentally desirable.

In one attempt to release a portion of the phytate P present in maizeand soybean meal the feed industry has added microbial phytase to thefeed of animals. This method of dealing with phytate in the grainappears to partially decrease the phosphorus excreted by the animal.This research apparently led to further methods of degrading phytate infeed. One method includes adding an enzymatic cocktail and Aspergillusniger mycelium to feed. These components function to hydrolyze phytatepresent in the corn-soybean diet. Turkeys fed the enzymatic cocktail andthe fungal mycellium showed enhanced performance and retention of P andCa. These feed studies were planned to dephosphorylate the corn andsoybean based feeds prior to consumption by the animal and thus reducethe P excreted. This method of dealing with phytate in the grain has thedistinct disadvantage of adding labor and cost to the feed.

Mogen, in U.S. Pat. No. 5,593,963, describes production of a temperaturestable phytase enzyme from Aspergillus in a corn or soy seed throughgenetic engineering techniques. The genetically produced phytase wasdesigned to reduce the phytic acid content in animal feed by degradingthe phytic acid being released from the grain and thus decrease thelevel of phosphorus excreted by the animal.

Low phytic acid mutant yellow dent corn seeds have been produced byRaboy and described in U.S. Pat. No. 5,689,054. This patent describesthe discovery of a single gene, nonlethal Ipa1 mutants in maize thatcause the reduction of kernel phytic acid phosphorus by up to 95% overthe wildtype phytic acid phosphorus levels. Raboy notes that while themutants of his invention are phenotypically very similar to thewild-type, the mutants would need to be introduced in to a breedingprogram in order to introduce the low phytic acid trait in to acommercial line. Moreover, Raboy explains that the low phytic acid maizemutants of his invention are characterized by a small kernel dry weightreduction which could result in a reductionin productivity and thathomozygous mutants may reduce or eliminate agronomically importantcharacteristics. As Raboy et al (Journal of Heredity 1989) has indicatedthat divergent selection for high protein consistently produces higherphytic acid lines, it is unclear how the Ipa1-R and Ipa2-R mutationsdescribed in the Raboy patent in yellow dent corn will interact withgenes for high-protein and oil-burdened corn seed. Thus one could nothave predicted with certainty whether it would have been possible tomaintain a high-protein oil burdened seed in combination with a lowphytic acid mutant.

Although the feed industry has addressed both the need for more energyin the feed and the need for less phytate-phosphorus, the feed industryhas not addressed the need for a method of providing, in a costefficient manner, both the high nutrient density (i.e., high protein andhigh oil) and the low phytic acid in feed. There is a need to reduce theamount of phytate salts formed in feed and increase the amount of energyin feed without having to add phytase and liquid oil to feed. Thereremains a need, which has not been addressed, for a grain having acombination of increased protein and oil burden and low phytic acidlevels. To reduce feed costs in animal production requires anutritionally dense material that is cost-effective and environmentallyfriendly. Additionally, there remains a need for a feed containing anoil burdened, protein laden corn with low phytic acid levels which canbe used for milling or for feed purposes.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a nutrient-dense grainthat contains both high levels of energy, through oil and improvedamino-acid content, through protein, and low levels of phytic acid.

Another object of the present invention is to decrease the phosphateand/or phosphorus excretion of animals consuming the feed whileincreasing the energy levels per daily feed intake and bioavailabilityof minerals and other nutrients.

An object of the present invention is to provide an animal feed thatcontains both high levels of energy through oil and protein and lowlevels of phytic acid.

Still a further object of the present invention is to provide a highlynutrient dense feed source to livestock which has less phytic acidpresent then the same feed source when made with regular commodity corn(i.e., no. 2 yellow dent corns).

Still a further object of the present invention is to provide a highenergy feed source to livestock containing sufficient supplies of anyrate limiting amino acid which has less phytic acid present then thesame feed source when made with regular commodity corn (no. 2 yellowdent corns).

It is another object of the invention to provide a method of reducinganimal phosphorus waste and/or pollution, and subsequent algal andmicrobial blooms caused therefrom, which method includes feedinganimals, such as pigs and chickens, the animal feed of the presentinvention.

In one embodiment, the present invention provides a non-lethal, mutantseed or grain of a cereal plant species, such as corn (maize), rice,barley and soy, having at least about 5% by dry weight, preferably atleast about 6%, alternatively at least about 7%, oil; at least 11% bydry weight, preferably at least about 12%, alternatively at least about13%, protein; and at least about a one third (33%) reduction in dryweight in the phytic acid amount (as measured by any of totalphosphorus, phytic acid or phytic acid phosphorus), preferably at leastabout a one half (50%) reduction, alternatively at least about 60-70%reduction, relative to wild-type seed of said species. Where the seed ofthe present invention is corn, the comparison in reduction is preferablymade relative to standard number (no.) 2 yellow dent corn.

In another embodiment, the present invention provides an increase inphosphorus availability of from 28% for yellow dent corn to greater thanabout 70%, preferably less than about 90%, alternatively about 80% toabout 84-85%. Availability being the amount of utilizable phosphoruscompared to total phosphorous from feed. The hybrid grain of the presentinvention is preferably a cross between useful inbreds and an inbredline ExSeed line U095-Ipa1-E (alternatively referred to as U095-E orU095py; deposited as strain designation EX1965py on Jul. 7, 1998 withAmerican Type Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209 USA, under conditions of the Budapest Treaty, Accession No.______. Source U095-py 1656-W97—Florida—100) The “E” or “py” designationused herein indicates the introduction of a Ipa1 mutation by the presentinventors. A number of other crosses and inbreds can be employed. Forexample, the following female inbreds BD68py, TR306py, WD22py andTR329py were crossed with male inbreds U095py, UU01py, UE95py, TR335pyand TR386py to make high-yielding hybrid combinations. Crosses withU095py are particularly preferred and the inbred U095py and hybrids madetherefrom are specific embodiments of the present invention. The hybridgrain of the present invention characterized by having ˜6% oil and 12%protein (or 3% more oil and 3% more protein than yellow dent corn) andat least about 33% reduction in phytic acid content.

In another embodiment, the present invention provides a feed containinga seed, as described herein, and at least one source of vitamins orminerals, containing, for example, any one or a mixture of at least twoof calcium or phosphorus or salts thereof, vitamin A, vitamin D, vitaminE, B₁₂, riboflavin, pantothenic acid, niacin, biotin, and traceminerals, such as iron, copper, manganese, zinc, iodine, and selenium,and/or additional feed additives, such as antibiotics, arsencials,chemotherapeutics, flavoring, antioxidants and plant extracts; said feedproviding a nutritionally balanced diet and a greater amount ofbiologically useful phosphorus to an animal consuming said feed thandoes the same feed formed with wild-type seed of the species. The feedof the present invention may also contain amino acid additives, such aslysine and methionine.

In another embodiment, the present invention provides an improved feedwhich is otherwise formulated for swine or poultry but includes theseed, preferably corn seed, of the present invention.

In yet another embodiment, the present invention provides a method ofincreasing bioavailability of phosphorus from products containingwild-type seed of a species, said method including the steps ofproviding a seed containing product, such as a feed as described herein,for consumption, wherein the seed containing product contains a seed ofthe present invention, and feeding the seed containing product to ananimal which will benefit from an increased bioavailability ofphosphorus.

In a further embodiment, the present invention provides germplasm whichwill yield the seed of the present invention. In a preferred embodiment,the present invention provides corn germplasm which will yield the cornseed described herein.

In yet another embodiment, the present invention provides a plantproduced from a seed of the present invention.

In yet a further embodiment, the present invention provides a seed ofthe present invention which is fully mature.

Still, further objects and advantages will become apparent from aconsideration of the ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a frequency distribution of phytate contents of screened M2seed.

DETAILED DESCRIPTION

The present invention provide grain, feed made from the grain, petfoodmade from the grain, and food products made from the grain. The grain ispreferably maize grain with the following characteristics: oil burdened,elevated protein content, and low phytate levels. The preferred grainhas at least 5% oil, at least 11% protein, and at least about 20% toabout 70%, preferably at least about to 33% to about 60% reduction inthe phytic acid level relative to wild-type grain, such as standardyellow dent corn. More preferably the grain has at least 6%, morepreferably 7% oil, at least 12%, and more preferably 13% protein, and atleast a one half reduction in the phytic acid level relative to standardyellow dent corn wherein the grain is low phytate. Percentages areexpressed on a dry weight basis as amount of a constituent per kernel,unless described otherwise. The combination of oil burdened, proteinladen, decreased phytate characteristics in grain makes a grain thatprovides more calories, protein and phosphorus and other nutrients tothe feeding animal. Pet foods, animal feeds and corn food products madeof the present invention will provide increased nutrition because of theincreased bioavailability of the components of the grain.

In other words the present invention includes an animal feed for aspecific animal type. In one embodiment, the present invention providesa feed having a gross energy and at least the same ratio of performancelimiting amino acids to gross energy as a nutritionally balanced feedusing no. 2 yellow dent corn formulated for a similar type animal. Thefeed is formulated with an energy source including elevated oil, proteinand low phytic acid maize. Additionally, the feed can contain at leastone protein source including a potentially performance limiting aminoacid component in a ratio to said gross energy such that the amino acidis not performance limiting, at least one source of vitamins andminerals; wherein the feed provides to the animal a higher calorie andlower phytic acid grain than no. 2 yellow dent corn in a nutritionallybalanced feed. The limiting amino acid can be various different aminoacids according to the needs of the animal species but it preferablyincludes lysine, tryptophan, threonine and methionine. The proteinsource of the feed can also include soybeans as a component. The feed ofthe present invention can also include the vitamins and a mineral sourcesuch as calcium, phosphorus and salt. In one embodiment, the feed of thepresent invention has vitamins and mineral sources which include any oneor a mixture of vitamin A, E, D, B₁₂, riboflavin, pantothenic acid,niacin, biotin; trace minerals, such as any one or a mixture of iron,copper, manganese, zinc, iodine, selenium, and feed additives, such asare known in the art and may include any one or a mixture ofantibiotics, arsanicals, chemotherapeutics, flavoring, antioxidants andplant extracts.

The present invention provides a method of increasing bioavailability ofphosphorus from maize containing products comprising the steps of:providing a maize containing product for consumption, wherein said maizecontaining product is formed from maize grain of the present invention,such as that characterized by having at least 5% oil, at least 11%protein, and at least a one third reduction in the phytic acid levelrelative to standard yellow dent corn, wherein the grain of the presentinvention is lower in phytate concentration than standard yellow dentcorn; and consuming said maize containing product which contains lessphytate in the maize material than the same maize product made withyellow dent corn wherein the bioavailability of the phosphorus in saidmaize is increased over the same product made with yellow dent corn.

The present invention further provides a feed for any non ruminantanimals. The feed of the present invention is particularly well-suitedas a constituent in the diets of swine or poultry.

In another embodiment, the present invention provides an animal feedhaving a gross energy content for a specific animal type, said animalrequiring a certain level of an amino acid in the feed to achieve goodperformance from said feed, the feed containing corn having an elevatedamount of oil compared to the average oil levels of no.2 yellow dentcorn and low phytic acid levels compared to the average phytic acidlevels of no. 2 yellow dent corn; a protein source, preferablysubstantially provided from the grain of the present invention; andhaving at least the same ratio of performance limiting amino acid togross energy as a nutritionally balanced feed using no. 2 yellow dentcorn formulated for a similar type animal.

The present invention provides grain having increased energy, proteinand low phytic acid. This grain can be used as feedstuffs for animals orthis grain can be milled. The present invention provides a new maizeseed, plant and grain that carry the oil burdened, protein laden and thelow phytic acid and the use for such new grain. The grain-basedfeedstuffs provide improved animal nutrition, and reduce theenvironmental impact of animal production. Even more particularly thisinvention provides an animal feed formulated using the grain of thepresent invention.

Many crop plants are used for the production of food for human or animalconsumption, for commercial processes yielding products for humanconsumption, for the development of industrial products and for otherpurposes. Traditionally, the improvement of crop plant species involvesthe introduction of desired traits by genetic crosses. The presentinvention likewise can be made repeatedly though the use of standardcrop, such as corn, breeding and mutation practices.

Corn grain is considered to be a high quality grain for use in foods andfeeds. High oil corn is considered more energy dense then other corn.High oil corn presently is commercially available from Dupont. However,this corn like all corn contains phytic acid. Until the presentinvention low phytic acid and oil burdened, protein laden corn plantsand grain did not exist. The invention also provides an improved flourfrom milling of the seed of the present invention. Thus, low phytate,oil burdened, elevated protein maize grain should address the need forenergy, protein dense corn and mineral bioavailability within the cerealgrain.

The method of repeatedly making the grain of the present invention is asfollows. Generally, in the course of a maize breeding program oilburdened, protein laden corn plants are crossed with maize plantscarrying the low phytic acid allele. Oil burdened corn plants can bedeveloped by recurrent selection as evidenced by University of Illinoishigh oil corn (commercially available from Dupont), or fromtransformation (methods of transforming corn are well known to thosehaving ordinary skill in the art). This is one part of the startingmaterial to make the present invention. The other part is a low phyticacid plant. Although this is not commercially available, experimentalmaterial is available.

High oil grain is commercially available from Dupont as either a TopCross grain or a high oil hybrid corn (high oil is any grain of cornhaving greater then 3.5% oil). Oil burdened corn is defined as cornhaving on average a higher percentage of total oil levels than theaverage standard yellow dent corn oil levels.

The following table shows the levels weight % of total oil of variousoils of a number of oilseed crops. The numbers under corn are the levelsin standard no.2 dent corn. TABLE 1 Fatty Acid Compositions ofCommercial Crops sun(hiO) canol lolinCan Crambe Corn Soy (C16:0) 6 4 5 211 11 palmitic (C16:1) 0.3 0.3 0.4 0.3 palmitoleic (C18:0) 5 1.5 1.5 1 24 stearic (C18:1) 20 (81) 62 62 17 28 22 oleic (C18:2) 68 (9)  20 27 858 53 linoleic (C18:3) 0.2 10 2 7 1 8 linolenicsun(hiO)—sunflower high oilcanol—canolalolinCan—low linoleic canolaCrambecornsoy—soybean

Thus the present invention encompasses grain that is oil burdened,preferably with fatty acid compositions in the ratios listed above. Thepresent invention also encompasses corn that has increased the fattyacid compositions such that there is not an overall increase but thereis a result increase in usefulness of the material to the animal fed thealtered corn. The present invention also encompasses grain that is oilburdened and the fatty acid compositions are in a different ratio thanlisted above.

The ratio of oils present in grain cereals can be varied though geneticmutation, selective breeding, transformation and the like. The essentialgrain for the feed can be produced by standard methods of production ofhybrid material such as the crossing of a oil burdened, elevated proteininbred with a low phytic acid inbred to produce hybrids having the graincharacteristics including the low fatty acid and the elevated oil.

As noted above, Raboy et al have reported a positive correlation betweenprotein selection and increased levels of phytic acid in corn. Thefollowing table lists the value of the amino acids present in one typeof nutrient dense corn which can be used to form the grain of thepresent invention. The protein in corn is controlled by many differentgenes. The preferred high protein corn has an increased percentage ofmost of the amino acids present in corn grain. One example of highprotein material has been the high protein corn developed by recurrentselection in the University of Illinois. Many of the other high proteincorn available have an increase in lysine and not necessarily an overallincrease in most amino acids. This type of high protein is also usefulas this increased protein is needed by the feeding animal for overallhealth and this protein is better utilized when combined with the othertraits of the present invention. To facilitate the breeding of hybridseed the high protein trait is additive or dominant. Quality proteinmaize includes the high protein material available from Wilson seeds andfrom Crows and from public depositories and universities. Alternatively,high protein material can be generated by recurrent selection or by theuse of certain mutation in corn like o2:su2 which have 49% more lysinethen normal hybrids but have decreased protein yields. In development ofthe present invention a high protein trait that was additive andincreased most protein levels beyond average yellow dent corn was used.Protein laden corn shall refer to corn that has increased amino acidcontent when compared to average amino acid content of yellow dent corn,and/or differing ratios of amino acid contents when compared to averageamino acid content of yellow dent corn. High protein corn shall refer tocorn that has increased amino acid content when compared to averageamino acid content of yellow dent corn. TABLE 2 Yellow-dent vs. EX404(EX404 has ˜1% wt % more oil and ˜2-3% wt % more protein then doesstandard yellow dent corn (average 3-3.5% by weight oil and 7-9% byweight protein) EX404 and ES404 as used herein are the same) Amino AcidComposition: Dent ES404 % change TRYPTOPHAN .06 .06 100% ASPARTIC ACID.57 .83 146% THREONINE .29 .47 162% SERINE .41 .63 154% GLUTAMIC ACID1.52 1.89 124% PROLINE .74 1.04 141% GLYCINE .33 .41 124% ALANINE .63.87 138% CYSTEINE .18 .24 133% VALINE .38 .47 124% METHIONINE .17 .26152% ISOLEUCINE .26 .36 138% LEUCINE 1.01 1.48 146% HISTIDINE .24 .35145% LYSINE .24 .31 129% ARGININE .29 .42 107%

This table clearly evidences that most of the the amino acid values ofthe nutrient dense corn are increased over the yellow dent material.This extra protein appears to be available for the animal's use when thephytic acid is substantially reduced. The ratio of proteins present ingrain cereals can be varied though genetic mutation, selective breeding,transformation and the like. The present invention provides a graincontaining increased levels of bioavailable protein in conjunction withincreased levels of oil and reduced levels of phytic acid.

Prior to the release by the USDA of the Low Phytic Acid mutants B73Ipa1-R, A632 Ipa1-R and selection thereof, there was only theconventional method of breeding for producing low phytic acid in cornseed. Low phytic acid in corn seed developed by standard breedingappeared to carry some undesirable agronomic traits. Due to therecessive nature of the phytic acid gene the preferred method requiresthat this gene be fixed in both inbreds. The mutant containing lowphytic acid can be developed according to the following method.Selection for phytic levels must be carefully performed as too low alevel of phytic acid may result in lethal seeds.

The low phytic acid plant of the present invention can be developed byfollowing the listed steps which do not take undue experimentation andcan be done by the ordinarily skilled person in the art of plantbreeding. The best method for generating a low phytic acid maize plantemploys maize pollen mutagenesis. The induced mutation in a haploidpollen grain would give rise to a heterozygous genotype in the seed.Since low phytic acid presently is known as a recessive gene, theresultant mutant seed must be planted and selfed and the resultantplants seeds assayed for the mutant phenotype. The assaying of the seedshould be done when the seed is in the mature stage or harvesting stage.

Mutagenesis is effected by conventional means in the art such asirradiation, chemical treatment, and transposable element insertion. Onestandard procedure is taught by Neuffer et al. Maize for BiologicalResearch, W. F. Sheridan (Ed.), Plant Molecular Biology Association(1982). This procedure uses ethyl methane sulfonate (EMS) applied topollen. The pollen is used for pollination and the resultant seeds areplanted and the seeds from the second generation can be tested forphytic acid content. The test for P has been known in the art since 1990when the HVPE method was published in Maydica 35:383 (1990) by Raboy.The method relies on differential migration of phosphorus compounds.After electrophoretically fractionating the compounds a chromatogramallows a semi quantitative assessment of the phytic acid relative toother compounds. An alternative method involves screening for higherlevels of inorganic P in the grain. For example grain samples can beground (to pass a 2 mm screen in a Wiley mill) followed by addition ofeither 50 mg of grain germ or 1 gram of endosperm in 15 ml of 0.4 M HCLin 0.7 Na₂ SO₄. Phytic acid precipitates as an iron salt. Phosphorus inthe ferric phytate precipitates and total P are determined. Phytic acidP (mg) are converted to phytic acid by use of a conversion factor of3.5. These results lead to the selection of the desired maize plantscontaining the desired alleles. Other methods of testing for P are knownand can be used to select plants. The seed containing the desired phyticacid is then increased. This process was employed in the presentinvention and inbred line were selected that carried new alleles at theIpa1 locus. These included EX404 (low phytic) which was crossed to oneof the inbreds of the present invention to form a hybrid that producedthe grain of the present invention. Additionally, the developed inbredsof the present invention were from stiff stalk, Lancaster and anotherversatile heterotic patterns so that the inbreds when crossed togetherwith the appropriate heterotic groups formed excellent hybrid material.It was also discovered that a number of the developed mutations of thepresent invention, though low in phytic acid were not the same mutant asthe Ipa1-R mutation as indicated by allelic testing.

The method used in the present invention can be used to form two inbredswhich would be crossed to form a high yielding hybrid. Inbreds arecommercially available from Universities and Foundation Seed Companiesand can be made by plant breeders skilled in the art of breeding maizeinbred lines. One or both of the inbreds fixed for the low phytic acidcan be crossed to an inbred which carries the elevated oil trait. Eitherinbred can cary the high protein trait if it was selected as an additivetrait. Thus allowing the hybrid combination to carry both traits therecessive low phytic acid and the dominant or additive oil genes. A cornplant is repeatedly bred until the low phytic acid allele is present intwo inbreds that cross well to one another. Likewise at least one of theinbreds in the hybrid combination must contain the oil burdened traitand/or the protein laden corn alleles.

Alternatively this grain can be produced by using the crossing method ofbreeding taught in the Dupont patent application WO92/08341 (U.S. Pat.No. 615,839). This method requires that the male pollinating plantcarries the oil burdened trait. In Dupont's method the oil pollinatorwhich is an inbred is mixed in with hybrid seed that is male sterile.The male pollinator then forms oil burdened grain on a sturdy hybridplant.

In the present invention the male pollinator would preferably have twotraits, a fixed recessive gene for phytic acid and the oil burdened,protein laden trait. Additionally, the male sterile hybrid would havethe low phytic acid trait also.

Methods of formulating an animal feed that contains additional energy,protein and low phytic acid in one grain source are herein described.The preferred method is to improve feeds or petfoods by substituting thegrain of the present invention for the corn grain normally employed.Other ingredients in the feed can be milling coproducts, proteiningredients, soybean meal, trace minerals and vitamins, and othercereals and feed additives and flavorings. This feed requires that grainbe produced that is characterized as having increased oil and increasedprotein/amino acid and low in phytic acid. Surprisingly, the combinationof the low phytic acid with the oil burdened and elevated proteinappears to allow the animal to utilize the extra protein that is in theoil burdened corn and the newly available phosphorus more efficientlyleading to increased feed efficiencies. The grain resulting from thishybrid combination can be used in an animal feed. This feed shouldcontain the combination of oil burdened, elevated protein and low phyticacid and other feedstuffs employed in the diets of either swine orpoultry.

Preferably if other grains are used, these grains are selected to haveas low a phytic acid content as is possible in that type of grain.Though the percentage of the grain as a part of the overall diet woulddetermine how stringently the selection against phytic acid would haveto be.

The present invention is of particular use for feeding non ruminantanimals. The feed ingredients for the standard swine diet are providedbelow. The feed mixture of the present invention substitutes the grainof the present invention for the no. 2 corn. The grain havingcharacteristics of low phytic acid and higher oil and increased aminoacids. This grain, when provided to the animal, results in a highercaloric density available and a greater bioavailability of a number ofnutrients such as minerals. The use of this grain in feed reduces theneed for added animal fat or vegetable oils.

Swine are primarily fed a fortified corn-soybean meal diet. Piglets,pre-weaning or early weaned, often have additives in the diets such asdried milk products, and other high protein and fat sources added to thediet. A complete swine diet provides all the nutritional needs of thepig in one diet. These diets may be prepared by mixing a balancedsupplement with corn: soybeans meal and a vitamin-mineral premix withcorn. Conventional feeds were often supplemented with sources ofsupplemental fat which are plant seed oils (extracted), grease andtallow, and commercial dry fat, corn oil, soybean oil or full fattedcooked soybeans. Flowability of the diet in automated feeders wascompromised at 6 percent fat. Overall 1 percent fat producedapproximately 2 percent improvement in efficiency (additional weightgain per weight of feed consumed). Increased oil from the use of thepresent feed (employing maize grain that is elevated oil and low phyticacid) increases the caloric density of the diet bioavailability ofmetals and reduces undesired waste. The feed of the present inventionemploys the corn grain of the present invention as a substitute for thegrain presently employed in diets for animals. The following two newfeed combinations will employ such grain. Both of these feed uses arefor animals which are preferable non ruminant animals. Such animalsinclude swine, poultry, cats, dogs, horses, sheep and the like.

Dietary nutrient density, when increased, maximizes animal performance.This includes animals such as sheep, swine, poultry, dogs, cats andhorses. This grain is primarily effective with most monogastric animals.To formulate a diet containing the grain of the present invention thatcontains higher levels of oil and amino acid than standard corn requiresthe step of substituting the grain of the present invention for theyellow dent corn (or even for the oil burdened corn) in the diet. Theinvention then however, requires the step of adjusting the protein toenergy level of the diet according to the needs of the animal. Forexample if the diet is for swine then the ratio of lysine should beincreased. If the diet is for poultry then the rate of methionine shouldbe increased and likewise for each animal.

The amino acid composition of protein specifically lysine which acts asa growth limiting amino acid for swine is increased in light ofincreased energy levels to keep a good balance of energy verses proteinwhen using the feed of the present invention. The invention itselfincreases the lysine. In the example given in table 2 of one of themaize grains containing increased oil and amino acid content theincrease in Lysine is 29% over the regular maize grain. If the increase.in the rate limiting amino acid in the grain of the present invention issufficient, the diet may not require additional increases in this aminoacid. However, to the extent that the grain does not carry all of thenecessary amino acids if sufficient amounts to put the diet in balance,they must be added to the diet.

The following table includes ingredients that are used in a standardfeed for swine. In the present invention these ingredients can likewisebe used, however, the standard corn and corn products made from standardcorn should be substituted by using the grain of the present inventionor products made from the grain of the present invention. Preferablyadded fats are not required in the feed. Additionally use of low phyticlegumes and other protein sources are encouraged. These examples, asothers herein, are illustrative of the use of the present invention andare not intended to limit the scope of the disclosed invention. Energy,Fat, Protein of Typical Ingredients Used in Swine Diets kcalmetabolizable Ingredient energy/lb protein % fat % Animal fat 3585 100Alfalfa 775 17 3.0 Barley 1380 11 1.8 Blood meal 1060 86.0 1.0 Corn,yellow 1555 8.0 3.25 Corn, gluten (ml) 1760 60.0 1.0 cottonseed meal1160 41.0 2.0 distillers dried 1515 27.0 8 grain w/solubles fish meal1500 60.0 6.0 Lysinehydrochloride, 1100 48.0 5.0 limestone, dicalciumphosphate Milo 1490 9.0 2.0 Molasses 910 3.0 Oats 1243 12 3.0 SkimmedMilk 1630 33 1.0 (dried) Soybean meal 1535 47.5 1.0 Soybeans (fat 165037 18.0 cooked) Soybean Oil 3300 100.0 sunflower 1000 28.0 2.0 Wheat1500 13 3.0 Whey 1405 14 .5

As is clearly evidenced in articles such as Adams, K. L. and Jensen A.H. “High Fat Maize in Diets for Pigs and Sows in Animal Feed” in Scienceand Technology, 17 (1987) 201-212, hogs fed higher energy diets usingjust high oil corn (and not the present invention of oil burdened,elevated protein and low phytic acid) with an energy: lysine ratioadjusted to the animal type (ie farrowing, starter pigs, finishing pigs)showed better feed efficiency then the same type of pig on a No.2 cornsoybean diet. The present invention used on this type of pig willprovide a diet that is not only calorie dense and thus increases feedefficiency but also is low in phytic acid which permits the feedinganimals to absorb from the grain the trace minerals, amino acids andmacro minerals needed for increased efficiency and gain. However, likethe previous tests with high oil corn the pig on a diet including grainof the present invention will require the increase in nutrients from theoil burdened, elevated protein and low phytic acid grain to be balancedwith an increase in the lysine level, if necessary.

Pig diets to be nutritionally balanced should have protein, amino acids,such as lysine, threonine, tryptophan, and major minerals, such ascalcium, phosphorus, salt, and vitamins such as A, D, E, B₁₂,Riboflavin, Pantothenic acid, Niacin, Biotin and trace minerals such asiron, copper, manganese zinc, iodine, selenium. Additionally feed dietsoften include feed additives such as flavorings and may have some of thefollowing chemicals added Apramycin, bacitracin, chlortetracycline,bambemycin, carbadox, lincomycin, penicillin, tylosin, virginiamycin andthe like.

Another potential use (other than as petfood and feed for swine) of thefeed described herein is for poultry feed. Poultry includes mostdomesticated fowl including duck, geese, pheasant, turkeys and chickens.The nutrient requirements of poultry and swine differ but the basicingredients in their diets are very similar. Poultry utilizes cerealgrains and grain co-products, fats and carbohydrates in their diets. Thelargest part of the poultry diet is made up of cereal grain. These atleast include corn, wheat, oats, milo, barley. However, in accordancewith the present invention the preferred main grain ingredient is oilburdened, protein laden and low phytic acid corn. The present inventionenvisions that little to no corn is used in the feed of the presentinvention that is not the grain or a product of the grain of the presentinvention. In addition the feed of the present invention includesproteins which may come from peanut meal, soybean meal, cottonseed meal,fish byproducts, blood meal and poultry byproducts and the like. Thefeed of the present invention may also include minerals and vitaminsparticularly calcium as limestone or oyster shells and vitamins D₃,choline, menadione, A, E, B₁₂ and feed additives such as antioxidants,antibiotics. Like swine, poultry seem to have a specific amino acidthat, if deficient, will reduce the animal's performance on the feed.For poultry, the limiting amino acid is methionine while for swine thelimiting amino acid is lysine. Thus, when the present invention feed isformulated there must be an increase in the amount of methionine andgeneral protein to keep the desired protein gross energy ratio in thediet as compared to the same ratio in the diet formulated with no. 2grain maize. Again the present invention provides not only the nutrientdense feed but increases the bioavailability of the trace metals due tothe decrease in the chelating agent, phytic acid, resulting in anincrease in feed efficiency of poultry on the present invention.

Although the description above contains many specificities, these shouldnot be construed as limiting the scope of the invention but as merelyproviding illustrations of some of the presently preferred embodimentsof this invention. Various other embodiments and ramifications arepossible within its scope.

The Iowa State University extension office publication entitled “Lifecycle Swine Nutrition,” PM489, revised 1996 on pages 12, 15 and 20 showa number of diets for sows and boars under standard productionconditions. The following information shows that pig feeds and poultryfeeds for different animals life stages are know in the prior art. Thefollowing is an example of one diet for a pig at a given developmentstage.

EXAMPLE 1

A pig having a moderate, lean growth potential with the followingweights are defined by the following stage development numbers. Pounds6-8=stage 1, Pounds 8-13 =stage 2, Pounds 13-18=stage 3, Pounds18-26=stage 4, Pounds 26-37=stage 5, Pounds 37-51==stage 6, Pounds51-69=stage 7, Pounds 69-91=stage 8, Pounds 91-118=stage 9, Pounds118-150 =stage 10, Pounds 150-188=stage 11, Pounds 188-233=stage 12,Pounds 233-283=stage 13.

The pig diet developed herein was formulated for a pig in stage six. Astage six pig would require for every one thousand pounds of feed: 648.4lbs of corn, 320.0 lbs soybean meal, dehulled 8.50 lbs of limestone,15.25 lbs. dicalcium phosphate, 4.10 lbs salt, 1.65 lbs trace mineralpremix, 0.75 lbs Fat soluble vitamin premix, 0.65 lbs vitamin B premix,0.50 lbs Biotin and Folic acid premix, 0.20 lbs choline premix and anyfeed additives approved by FDA. This feed is made according to thepresent invention by substituting the corn of the present invention intothe feed instead of the no. 2 corn and increasing the level of lysine tomatch the increased energy in the diet to avoid the lack of lysine fromlimiting in the animal's growth. If the feed is pelleted then thevitamins should be increased approximately 20%.

Corn is the basic cereal grain used in animal feeds. Corn contains manyof the essential nutrients that are required by the animal. The modernanimal, with its tremendous genetic ability to deposit lean tissueversus fat, has a tremendous requirement for nutrients. As this demandfor fat, has a tremendous requirement for nutrients, as this demand forincreased nutrition, we have at the same time see a decline in feedintake, While animal genetics had and continue to improve carcassqualities by reducing total body fat, the environmentalists haveindicated that our concentrated animal feeding operations are pollutingthe ground and subsequent groundwater. Knowing that corn is the leadingfeeding ingredient used in animal production the present invention is acorn that contains concentrated nutrients, and nutrients that had higherbiological availability to the animal. Such nutrients include protein,energy, amino acids and minerals. The key mineral, phosphorus, isessential for growth, health and production. Phosphorus in corn is only˜30% available to the animal because of the phytic acid content of thecorn. However, the nutrient dense corn of the present invention has agreater availability of the phosphorus.

EXAMPLE 2

This experiment determines the response of commercial broiler chickensto low phytic acid mutant yellow dent corn as compared to yellow dentcorn.

The trials use commercial broiler chickens in a 13-day feeding study.The basal diet is an NRC (National Recommendation Committee) based corndiet. The treatment is a replacement of conventional yellow dent cornwith low phytic acid hybrid grain obtained from a cross between B73Ipa1-R and an Exseed inbred line UU01-Ipa1-E (alternatively referred toherein as UU01-E or UU01-py) which is a low phytic mutant of yellow dentcorn.

All experiments were managed under the Good Laboratory Practices withanimals welfare at the forefront. Feed and water is supplied ad libitum,body weight and feed intake is measured throughout the experiments,phosphorus availability is measured by means known in the art. Carcassevaluations, total body weight gain, feed conversion ratio, mortality,morbidity and intakes are reported. The expected results are improvedphosphorus availability of low phytic acid yellow dent corn versusyellow dent corn.

It is believed that the grain of the present invention in the diet willresult in less excreted phosphorus. This will result in less pollution.

In this study, 8 day old New Hampshire×Columbian male chicks with anaverage initial weight of 73.7 g were used to compare the phosphorusavailability of low phytate yellow dent corn of the present inventioncompared to conventional corn (yellow dent). This experiment was a CRD;7T×4R×5C, 13 day trial (7 treatments, 4 replications, 5 chicks pertreatment).

The basal diet was as follows: cornstarch/dextrose (2:1 ratio) to 100%;soybean meal 47.4%; soybean oil 5%; Limestone 1.2%; Salt 0.35%; vitaminmix (A, D₃, E, K, scotin, riboflavin, pantothenic acid, niacin, cholinechloride, folic acid, thiamine, B₆, B₁₂) 0.35%; mineral mix (copper,iodine, iron, manganese, selenium, zinc) 0.15%; choline 60, 0.1%; DL-Met0.25; flavomycin 0.05%. The basal diet contained 0.1% availablephosphorus, 0.63% Ca and 23% CP. All percentages are on a weight basis.

The treatment design for this trial was as follows 1: Basal diet; 2:Basal plus 0.06% phosphorus (inorganic phosphorus) from potassiumphosphate (KH₂PO₄); 3: Basal plus 0.12% phosphorus from potassiumphosphate (KH₂PO₄); 4: Basal plus 20% conventional corn; 5: Basal plus40% conventional corn; 6: Basal plus 20% low phytate corn; 7: Basal plus40% low phytate corn. Weight gain - 8-20 d (g) 1 2 3 4 5 6 7 R1 227.8269.8 291.2 209.2 249.4 253.2 283.0 R2 216.8* 267.6 295.4 224.0 258.8249.4 273.0* R3 237.0 285.6 294.4 229.8 259.6 244.2 255.0 R4 224.6 258.4300.2 232.8 236.8 241.6 269.6 mean 226.5^(d) 270.3^(b) 295.3^(a)223.9^(d) 251.1^(c) 247.1^(c) 270.1^(b) g/c/d  17.4  20.7  22.7  17.2 19.3  19.0  20.7Pooled SEM = 4.6;LSD = 13.5;*chick removed or died;g/c/d = grams/chick/day

Feed Intake - 8-20 d (g) 1 2 3 4 5 6 7 R1 346.4 383.2 417.8 317.0 354.2358.8 367.8 R2 325.9* 397.6 428.4 332.2 368.6 350.6 365.6* R3 353.0415.4 414.6 337.6 371.0 338.4 355.6 R4 348.6 387.0 435.6 343.8 349.8344.6 367.6 mean 343.4^(d) 395.8^(b) 424.1^(a) 332.6^(c) 360.9^(cd)348.1^(de) 364.1^(c) g/c/d  26.4  30.4  32.6  25.5  27.7  26.7  28.0Pooled SEM = 5.3; LSD = 15.6;*chick removed or died

Gain/Feed - 8-20 d (g) 1 2 3 4 5 6 7 R1 0.658 0.704 0.697 0.660 0.7040.706 0.769 R2 0.665* 0.673 0.690 0.674 0.702 0.711 0.747* R3 0.6710.688 0.710 0.681 0.700 0.722 0.717 R4 0.644 0.668 0.689 0.677 0.6770.701 0.733 mean 0.659^(c) 0.683^(cd) 0.696^(bc) 0.673^(de) 0.695^(bc)0.710^(b) 0.741^(a)Pooled SEM = 0.006;LSD = 0.020;*chick removed or died

Tibia Bone Ash (%) 1 2 3 4 5 6 7 R1 30.7 35.8 37.1 29.4 34.2 31.5 36.5R2 30.2* 33.9 38.5 29.0 31.7 28.9 34.9* R3 28.1 33.3 36.9 28.4 32.5 31.134.1 R4 30.2 33.7 36.6 29.1 31.0 29.7 35.0 mean 29.8^(d) 34.1^(b)37.2^(b) 28.9^(d) 32.3^(c) 30.3^(d) 35.1^(b)Pooled SEM = 0.5;LSD = 1.5;*chick removed or died

Tibia Bone Ash (g) 1 2 3 4 5 6 7 R1 0.242 0.318 0.427 0.215 0.262 0.2830.338 R2 0.230* 0.312 0.418 0.232 0.296 0.250 0.345* R3 0.239 0.3270.394 0.233 0.270 0.253 0.327 R4 0.241 0.300 0.390 0.248 0.254 0.2610.339 mean 0.238^(c) 0.314^(c) 0.407^(a) 0.232^(c) 0.270^(d) 0.261^(d)0.337^(b)Pooled SEM = 0.006;LSD = 0.019;*chick removed or died

Slope ratio and standard curve analysis from the above results indicatesthat the low phytate corn contains 3 times more available phosphorusthan in conventional corn.

EXAMPLE 3

The method of producing these elite, agronomically sound and highyielding mutants is a known method called mutagenesis. The process isoutlined in the Neuffer paper Maize Genetic Newsletter 45:146. It shouldbe noted that EMS is a mutation process. Like all mutation processes theact of mutation can adversely effect the agronomic traits especiallyyields of the plant. However, the starting germplasm is superior to thatin which the phytate mutant was previously formed. Thus the overallagronomic traits of the plant of the present invention are more easilypreserved and selected for then the industries approach of recurrentselection or backcrossing. Mutations were induced in the inbred line bytreating pollen with ethyl methane sulfonate in paraffin oil accordingto the procedure described by Neuffer (1974). This treatment wasperformed on a number of inbreds from the various plant genotypes ofcereal. This example will focus on the development of maize low-phytatemutants by this process. This mutagenesis process has been used to makea number of cereal mutants.

The general steps of the process of the present invention includetreating inbred pollen (in this case maize) with ethyl methane sulfonatehereinafter “EMS”. Inbred pollen is placed in EMS in oil for minutes. Apaint brush is used and the pollen is brushed on to the silks of areceptive corn ear. This forms the Mutant-1 (M1) seed. Such seed aregrown and self-pollinated to produce the Mutant-2 (M2) kernels. Theresulting M2 kernels are tested for the low phytate phenotype.

EXAMPLE 4

The HVPE method is a common test for low phytate mutants. (Raboy, Mydica35:383 (1990)). The method relies on differential migration ofphosphorus compounds. After electrophoretically fractionating thecompounds a chromatogram allows a semi quantitative assessment of thephytic acid relative to other compounds. An alternative method isscreening for higher levels of inorganic P in the grain. For examplegrain samples can be ground (to pass a 2 mm screen in A Wiley mill) addeither 50 mg of grain germ or 1 gram of endosperm in 15 ml of 0.4 M HCLin 0.7 Na₂ SO₄. Phytic acid precipitates as an iron salt. Phosphorus inthe ferric phytate precipitates and total P are determined. Phytic acidP (mg) are converted to phytic acid by conversion factor 3.5.

The principles of phytate measurement are known. In the method employedin the present study, a solution of 5-sulfosalicylic acid and FeCl₃(Wade reagent) forms a pink chromophore. Phytic acid binds iron in thissolution decreasing the level of the pink color. The measurement of thisloss of color can be used as an indication of phytic acid levels. Sincethe blank contains no phytate, all readings of samples that do containphytate will be negative numbers. If there is too much phytate, however,the iron-phytate complex can precipitate as a milky white substance. Inthis case the pink color will not be present but the milky white matterwill absorb light and result in falsely high readings. Thus some visualobservation may be necessary. This may necessitate using a smalleraliquot (less than 25 microliters) of the corn extract if the cornvariety has high levels of phytate.

A rapid screening procedure, such as described as follows, may be usedto score for putative low phytate seeds. In this procedure, a singleedge razor is used to cut the kernel tip cap off just behind the blacklayer. The cut should transect the scutellum at a point at or near theradicle tip. Ususally, 8 representative kernels were selected from eachear. The kernels are then placed, cut surface up, on a microplate thathas the surface covered with cellophane tape (sticky side up). Thestaining procedure was completed after dissection of at least 100families.

Staining was done with the use of a repeating pipette to place a 10microliter drop of Wade reagent (as described below) on the cut surfaceof each kernel. After a few minutes the color disappeared as the phyticacid from the scutellum binds the iron in the Wade reagent. Observationswere made for families that segregate for slower disappearance of thepink color relative to the others (perhaps 5% of the total). Theseslower families were re-analyzed by the quantitative procedure describedherein.

Phytate was quantified as follows. Individual kernels (7 to 10 from eachfamily) were crushed in steel plate of a Carver hand-pump press (bestresults obtained when wells of plate lined with glycine paper andcrushed with about 5000 lbs. pressure) and placed into 1.5 mlmicrocentrifuge tubes. 1 ml of 0.65 N hydrochloric acid was added andallowed to stand overnight. The combination was mixed by inversion thenext day and allowed to settle for 5 minutes. A 15 microliter aliquot ofthe supernate was added to a microfuge tube with 100 microliters ofbuffer A (as described herein) and mixed. Low phytate mutants turn avery blue color due to the high phosphorus levels of the seeds. Mutantswere generally retested the following day. The 0.65 N HCl extractionsolution was made by adding 216 ml of 12.1 N HCl to 3784 ml water.Reagent A was made fresh daily and included 2 parts (by volume)deionized water, 1 part (by volume) ascorbic acid solution, 1 part (byvolume) ammonium molybdate solution and 1 part (by volume) H₂SO₄solution. Ammonium molybdate solution was made by adding 25 g (NH₄)₆MO₇O₂₄×4H₂O to make 1 L with water. H₂SO₄ solution was made by adding167 ml of 36 N sulfuric acid to 833 ml water. Ascorbic acid solution wasmade by adding 100 g L-ascorbic acid to make 1 L with water. Ascorbicacid solution was stable with refrigeration for about seven weeks butonly about two hours unrefrigerated.

Phytic Acid standards were prepared by means known in the art.

Maize kernel (M2) free phosphate was visually screened as follows.Kernels were selected, the phenotypes noted and placed in a multiplewell crushing plate. Kernels were crushed in the multiple well crushingplate using a hydraulic press. Crushed kernels were transferred into 1.5ml eppendorf microcentrifuge tubes. 0.5 ml of Reagent A was added. Afterallowing 2 hours reaction time, 0.5 ml reagent B was added. The tubeswere capped and mixed by inverting. The reactions were scored visuallyfor blueness after 1 hour, using a light box where necessary, and thebluest samples were selected as having highest phosphate. Often, thebluest samples from each family (ear) were selected and compared forfinal selection. Reagent A of this assay was prepared from 50 ml DMSOand 50 ml Reagent B. Reagent B was made fresh daily and prepared from 60ml distilled water; 30 ml of 10% ascorbic solution (10 g ascorbic acidwater to 100 ml total volume; the ascorbic solution was refrigerated andstable for 1 week); 30 ml of 3.5% ammonium molybdate solution (2.5 g(NH4)₆ Mo₇ O₂₄*4H₂O add water to 100 ml total volume); 30 ml of 6Nsulfuric acid solution (170 ml water plus 25 ml concentrated H₂SO₄,adjust to 200 ml total volume with water).

Phytate (Red Test) was quantitatively measured as follows. Approximately12 mature seeds were crushed in a steel crush plate of a Carverhand-pump press. Best results were obtained when wells of the plate werelined with weighing paper. Kernels were crushed with 5000 to 10000 lbs.pressure and transferred to Eppendorf tubes. 1 ml of 0.65 N HCl wasadded to same, allowed to sit overnight and mixed the following day bytube inversion. To assay, 200 μl of Wade-A reagent (described below) wascombined with 10 μl of the above obtained corn/HCl juice extract inindividual wells of a microtiter plate. Any change in color was notedand samples which remained red were noted as low in phytate sincephytate binds with iron and turns the solution white. Quantitation maybe completed with a spectrophotometer measuring at 490 nm. Wade-Areagent used herein was prepared by adding 25.4 g of 5-sulfosalicylicacid and 350 mg of FeCl₃.6H₂O (ground with mortar and pestel ifnecessary) to 1.5 L deionized water. NaOH was used to adjust the pH to3.05 and volume adjusted with d.H₂O to 2 L. This reagent was stable in arefrigerator for about 1 mo. 0.65N HCl was prepared by adding 216 ml HCl(12. IN) to 3784 ml of d.H₂O.

These assays allow the selection of the desired maize plants containingthe desired alleles. Other methods of testing for phosphorus are knownand can be used to select plants.

Seed containing the desired levels of phytic acid are then increased.This process was employed in the present invention and a number ofinbred lines were selected that carried the low phytic acid mutation.These included several low phytate inbred lines with good combiningability which were crossed together to form a hybrid that produced thegrain of the present invention. Thus, the developed inbreds of thepresent invention were produced from stiff stalk, Lancaster and anotherversatile heterotic patterns so that the inbreds when crossed togetherwith the appropriate heterotic pattern formed excellent hybrid material.It was also discovered that a number of the developed mutations of thepresent invention though low in phytic acid were not the same mutant asthe Ipa1-R mutation. Additionally, the seed were screened forgerminability in standard seed germination tests. It was found that somelow phytate mutants were unable to germinate whereas others wouldgerminate normally. Only the seed with good germination characteristicswere maintained.

FIG. 1 shows an example of some data obtained form 3 such inbred linesscreened for phytate content. Plotted as a frequency distribution curveit is clearly evident that there were a few samples with very lowphytate content (less than 0.5 units (weight percent) whereas the bulkof the samples were higher in phytate content. The line known as UO95line was selected as a starting material for its higher than averageprotein and oil levels. This line produces seed which are oil burdenedand protein laden and which germinate normally. UO95py retains thesecharacteristics with the low phytate levels described above. Whencrossed with certain other inbred lines, the resulting hybrid producesgrain which are oil burdened and protein laden and contain low phyticlevels.

The following table represents phytic acid contents (mg/g of seed) formutant corn according to the present invention compared with wild-typeseed. No. Wild-type Phytic Mutant Phytic % Seeds Line acid Line acidreduction 24 UU01 1.16 UU01-py 0.17 85.3 12 U095 1.85 UO95-py 0.14 92.412 WD22 1.45 WD22-py 0.05 96.6

The following provides an example of an inbred line according to thepresent invention. % phytate Inbred Protein Oil Phytate reductionWild-type UO95 13.4 606 1.85 UU01 12.7 2.9 1.16 B73 11.3 4.4 — WD22 — —1.45 Mutants UO95py 14.4 5.3 0.14 85.3 UU01py 12.2 3.1 0.17 92.4B73lpa1-R 13.2 3.2 — — WD22py — — 0.05 96.6Protein and oil contents were measured by NIR analysis on a Dickey-JohnReflectance Near Infra Red Spectrometer.

The grain of the present invention can also be used as a substitutesource for the corn grain or flour used to make corn tortilla, cornmeal, and cornflakes by substituting the grain of the present inventionin the recipe and baking or processing as one would normally.

The grain of the present invention can also be used as a substitute forthe corn wet milling industry by substituting the grain of the presentinvention in order to increase milling efficiency and recoverable starchcontent. Animal feed made as a by-product of the milling process is alsosubstantially reduced in phytate content.

The entire contents of references referred to herein are incorporated intheir entirety by reference.

1-18. (canceled)
 19. Seed of corn inbred line UO95py, representativeseed of said line having been deposited under ATCC Accession No. 203034.20. A hybrid corn seed produced by crossing corn inbred line UO95py witha second inbred corn line. 21-23. (canceled)
 24. A corn plant producedby growing the seed of claim
 19. 25. A corn plant produced by growingthe seed of claim 20.